Seminar Presentation

on
Microscopy
Department of Microbiology
SKIMS SOURA
 INTRODUCTION HISTORICAL BACKGROUND PROPERTIES OF MICROSCOPE
PARTS OF MICROSCOPE TYPES OF MICROSCOPE

Microscope
Derived from two Greek Words
 Micron means “Small”
 Scope means “Aim”

By definition a microscope
 An optical instrument consisting of a lens or combination of lenses to
produce magnified images of small objects especially which are too small
to be seen by naked eye.
INTRODUCTION HISTORICAL BACKGROUND PROPERTIES OF MICROSCOPE
PARTS OF MICROSCOPE TYPES OF MICROSCOPE
INTRODUCTION HISTORICAL BACKGROUND PROPERTIES OF MICROSCOPE
PARTS OF MICROSCOPE TYPES OF MICROSCOPE
INTRODUCTION HISTORICAL BACKGROUND PROPERTIES OF MICROSCOPE
PARTS OF MICROSCOPE TYPES OF MICROSCOPE
INTRODUCTION HISTORICAL BACKGROUND PROPERTIES OF MICROSCOPE
PARTS OF MICROSCOPE TYPES OF MICROSCOPE
 INTRODUCTION HISTORICAL BACKGROUND PROPERTIES OF MICROSCOPE
PARTS OF MICROSCOPE TYPES OF MICROSCOPE

 Magnification or magnifying power

• is the degree of increase in size of optical image over the actual size of object
being viewed.
• Total magnification of a field is the product of the magnification of objective
lens and ocular lens:

• Scanning field ( 40x)

• Low power field (100X)
• High power field (400x) and
• Oil immersion field (1000X)
 INTRODUCTION HISTORICAL BACKGROUND PROPERTIES OF MICROSCOPE
PARTS OF MICROSCOPE TYPES OF MICROSCOPE

 Resolution power:
• Ability to produce separate images of closely placed objects so that they can be
distinguished as two separate entities. The resolution

• Unaided human eye is about 0.2 mm (200 μm).

• Light microscope is about 0.2 μm.
• Electron microscope is about 0.5 nm (0.2 μm).
• Resolution depends on refractive index of the medium.
• Oil has a higher refractive index than air; hence, use of oil enhances the
resolution power of a microscope.
 INTRODUCTION HISTORICAL BACKGROUND PROPERTIES OF MICROSCOPE
PARTS OF MICROSCOPE TYPES OF MICROSCOPE

 Good contrast

 the difference in light intensity between the image and the adjacent background relative
to the overall background intensity.

 This can further be improved by staining the specimen. When the stains bind to the cells,
the contrast is increased

 This is achieved by use of lenses. There are two type of concave lenses used:
• Ocular lens with a magnification power of the 10X.
• Objective lens-
• scanning (4x), low power (10X),
• high power (40x) and oil immersion (100X).
INTRODUCTION HISTORICAL BACKGROUND PROPERTIES OF MICROSCOPE
PARTS OF MICROSCOPE TYPES OF MICROSCOPE
INTRODUCTION HISTORICAL BACKGROUND PROPERTIES OF MICROSCOPE
PARTS OF MICROSCOPE TYPES OF MICROSCOPE

 Bright-field OR Light Microscope

 Dark field Microscope
 Phase contrast Microscope
 Fluorescence Microscope
 Confocal Microscope
 Electron Microscope
 Inverted plane Microscope
INTRODUCTION HISTORICAL BACKGROUND PROPERTIES OF MICROSCOPE

PARTS OF MICROSCOPE TYPES OF MICROSCOPE

BRIGHT-FIELD
OR
LIGHT MICROSCOPE
INTRODUCTION HISTORICAL BACKGROUND PROPERTIES OF MICROSCOPE
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BRIGHT-FIELD

The bright-field or light microscope forms a dark image against a

brighter background.

Working Principle
The rays emitted from the light source pass through the iris
diaphragm and fall on the specimen. The light rays passing through
the specimen is gathered by the objective and a magnified image is
formed. The image is further magnified by the ocular lens to produce
the final magnified virtual image.
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BRIGHT-FIELD
 INTRODUCTION HISTORICAL BACKGROUND PROPERTIES OF MICROSCOPE
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BRIGHT-FIELD

Structure
The parts in a bright-field microscope are divided into three groups
Mechanical Parts.
 Base
 C-shaped arm
 Mechanical stage
Magnifying Parts
 Ocular lens
 Objective lens
Illuminating Parts
 Condenser:
 Iris diaphragm
 Light source
 Fine and coarse adjustment knobs
 INTRODUCTION HISTORICAL BACKGROUND PROPERTIES OF MICROSCOPE

PARTS OF MICROSCOPE TYPES OF MICROSCOPE

BRIGHT-FIELD

Applications
Bright-field microscope is used:-
• Viewing stained or naturally pigmented specimens
• Stained prepared slides of tissue sections or living photosynthetic organisms.
INTRODUCTION HISTORICAL BACKGROUND PROPERTIES OF MICROSCOPE

PARTS OF MICROSCOPE TYPES OF MICROSCOPE

DARK-FIELD
MICROSCOPE
 INTRODUCTION HISTORICAL BACKGROUND PROPERTIES OF MICROSCOPE

PARTS OF MICROSCOPE TYPES OF MICROSCOPE

DARK-FIELD

Dark field microscope, the object appears bright against a dark background.

 The dark field condenser has

 central opaque area that blocks Light from entering the objective lens directly.
 peripheral annular hollow area which allows the light to pass through and focus
on the specimen obliquely.
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DARK-FIELD

Ray Diagram of Dark field

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DARK-FIELD

Applications
• Identify the living unstained cells and
• Thin bacteria like spirochetes, the most notorious bacterium
Trepnonema pallidum_the causative agent for syphilis, which cannot
be visualized by light microscopy.

Trepnonema pallidum
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Phase contrast
Microscope
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Phase contrast

 This microscope visualizes the unstained living cells by creating difference in

contrast between the cells and water.

 It converts slight differences in refractive index and cell density into easily
detectable variations in light intensity.

 The background, formed by un deviated light is bright, while the unstained object
appears dark and well defined.
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Phase contrast
INTRODUCTION HISTORICAL BACKGROUND PROPERTIES OF MICROSCOPE

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Phase contrast

How phase contrast microscope works

 INTRODUCTION HISTORICAL BACKGROUND PROPERTIES OF MICROSCOPE

PARTS OF MICROSCOPE TYPES OF MICROSCOPE

Phase Contrast

Applications
Phase contrast microscopy is especially useful for studying:
 Microbial motility

 Determining the shape of living cells, and

 Detecting bacterial components, such as

 Endospores and
 Inclusion bodies which become clearly visible because they have refractive
indices markedly different from that of water.
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Phase contrast
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Fluorescence
Microscope
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Fluorescence Microscope

The "fluorescence microscope" refers to any microscope that uses

fluorescence property to generate an image.
Principle of Fluorescence Microscope
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Fluorescence Microscope

Applications
Epifluorescence microscope: it is the simplest format of
fluorescence microscope, which has the following
applications.
 Auto fluorescence: Certain microbes directly
fluoresce when placed wider uv lamp, e.g. Cyclospora
(a protozoanparasite).
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Fluorescence Microscope

• Microbes coated with fluorescent dye: Certain microbes fluoresce when they
are stained non-specifically by fluorochrome dyes.

• Acridine orange dye is used for the detection of malaria parasites by a method
called as quantitative buffy coat (QBC) examination.

• Auramine phenol is used for the detection of tuberclebacilli

 INTRODUCTION HISTORICAL BACKGROUND PROPERTIES OF MICROSCOPE

PARTS OF MICROSCOPE TYPES OF MICROSCOPE

Fluorescence Microscope

Tubercle bacilli seen under fluorescence

microscope
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Confocal Microscope

CONFOCAL
MICROSCOPE
 INTRODUCTION HISTORICAL BACKGROUND PROPERTIES OF MICROSCOPE

PARTS OF MICROSCOPE TYPES OF MICROSCOPE

Confocal Microscope

 Is an updated version of fluorescence microscopy.

 Confocal microscopy is an optical imaging technique for increasing optical resolution and
contrast of a micrograph.

 Uses pin hole screen to produce high resolution images.

 Eliminates out of focus

 So images have better contrast and are less hazy.

 a series of thin slices of the specimen are assemble is generate a three dimensional images.
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PARTS OF MICROSCOPE TYPES OF MICROSCOPE

Confocal Microscope

Principle
In confocal microscopy two pin holes are typically used :-
• a pin hole is placed in front of the illumination source to allow transmission only through a
small area
• This elimination pin hole is imaged on to the focal plane of the specimen that is only a point
of a specimen is eliminated at one time
• Fluorescence excited in this manner at the focal plane is imaged on to a confocal pinhole
placed right in front of the detector.
• Only fluorescence excited within the focal plane of the specimen will go through the detector
pinhole
• Scanning of small sections is done and joined them together for better view .
 INTRODUCTION HISTORICAL BACKGROUND PROPERTIES OF MICROSCOPE

PARTS OF MICROSCOPE TYPES OF MICROSCOPE

Confocal Microscope

Applications:
 Confocal microscopy allows analysis of fluorescent labelled thick specimen without
physical sectioning

 Three- Dimensional reconstruction of specimen.

 More color possibilities.

 Improved resolution.
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Confocal Microscope
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Electron
Microscope
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Electron Microscope

An electron microscope (EM) uses accelerated electrons as a source

of illumination.

Because the wavelength of electrons can be up to 100,000 times

shorter than that of visible light photons.
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Electron Microscope

• The EM has a much better resolving power than a light

microscope; hence, it can reveal the details of flagella, fimbriae
and intracellular structures of a cell.
• Electron microscopes are of two types:
1. Transmission electron microscope
2. Scanning electron microscope.
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Transmission Electron Microscope

The TEM is somewhat analogous to the brightfield light microscope in

terms of the way it functions. However, it uses an electron beam from
above the specimen that is focused using a magnetic lens (rather than a
glass lens) and projected through the specimen onto a detector.
Working Principle
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Transmission Electron Microscope

Applications
1. TEMs offer very powerful magnification and resolution.

2. TEMs have a wide-range of applications and can be utilized in a variety of

different and scientific, educational and industrial fields.

3. TEMs provides information on elements and compound structure.

4. Images are high-quality and detailed

INTRODUCTION HISTORICAL BACKGROUND PROPERTIES OF MICROSCOPE

PARTS OF MICROSCOPE TYPES OF MICROSCOPE

Transmission Electron Microscope

 INTRODUCTION HISTORICAL BACKGROUND PROPERTIES OF MICROSCOPE

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Scanning Electron Microscope (SEM)

 SEM uses electron beams to visualize surfaces; useful to observe the three-
dimensional surface details of specimens.

 Scanning electron microscopy is used for inspecting topographies of specimens at

very high magnifications.

 SEM magnifications can go to more than 300,000 X.

 INTRODUCTION HISTORICAL BACKGROUND PROPERTIES OF MICROSCOPE
PARTS OF MICROSCOPE TYPES OF MICROSCOPE

Scanning Electron Microscope (SEM)

Applications
 Detailed 3D and topographical imaging and the versatile information garnered
from different detectors.

 Instrument works very fast.

 Modern SEM allow for the generation of data in digital form.

 Can magnify up to 100,000x

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Scanning Electron Microscope (SEM)

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Scanning Tunneling
Microscope
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Scanning tunnelling microscope

 A scanning tunneling microscope (STM) is an instrument for imaging

surfaces at the atomic level.
 The scanning tunneling microscope was invented in 1982 by Binnig and Rohrer.

 The instrument consists of a sharp conducting tip which is scanned across a flat
conducting sample.
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Inverted Plane
Microscope
INTRODUCTION HISTORICAL BACKGROUND PROPERTIES OF MICROSCOPE

PARTS OF MICROSCOPE TYPES OF MICROSCOPE

Inverted Plane Microscopes

 INTRODUCTION HISTORICAL BACKGROUND PROPERTIES OF MICROSCOPE
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Inverted Plane Microscopes

An inverted microscope is a microscope with its light

source and condenser on the top, above the stage pointing
down, while the objectives and turret are below the stage
pointing up.
It was invented in 1850 by J. Lawrence Smith, a faculty
member of Tulane University (then named the Medical
College of Louisiana).
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Inverted Plane Microscopes

Applications
 Inverted microscopy is a very popular technique for live cell
imaging. Here, living cells are observed through the bottom of a cell
culture vessel.
 Inverted microscope is also used for visualization of
the mycobacterium tuberculosis bacteria in the technique called
microscopic observation drug susceptibility assay (MODS).